This invention relates to analytical methods wherein samples are extracted with organic solvents to obtain analytes free of unextracted sample constituents such as proteins, followed by specific protein binding assays for the extracted analytes. In particular, this invention relates to those methods in which substantially water immiscible organic solvents have been used in such extractions.
Specific protein binding assays are defined herein as methods for analysis which exploit the capacity of various proteins to reversibly bind other substances with varying degrees of specificity. Such assays are in general well known. Exemplary specific protein binding assays are the sandwich, sequential saturation, and competitive methods. Such assays usually employ an antiserum which contains antibodies capable of reversibly, specifically binding the substance to be determined, i.e., the analyte. However, naturally occurring, non-immune binding proteins such as intrinsic factor and thyroxine binding globulin have been used satisfactorily as specific binding proteins. The analytes which can be determined by specific protein binding assays are virtually limitless; if a non-immune protein specific for the analyte cannot be found then an antibody having the desired specificity can be raised by known techniques.
The method of this invention particularly relates to those analytes such as hormones and drugs, generally low molecular weight substances, which are soluble to at least some extent in organic solvents.
It has been conventional practice in specific protein binding assays for such analytes to extract the analyte-containing samples with organic solvents. This has been done primarily for three reasons. First, organic solvents are well-known protein denaturing agents. Since many analytes are noncovalently bound by endogenous body fluid proteins it has been considered desirable to release the analyte from the proteins so that it can be determined along with the proportion of analyte which is normally free. Organic solvents may achieve the desired release of analyte by denaturing the binding proteins.
Second, water immiscible solvent extraction has been useful to extract analytes from mixtures containing related compounds. For example, cortisol and estriol may be solvent extracted from their sulfate and glucuronide derivatives.
Third, water immiscible organic solvents have been employed to extract analytes found in samples at low concentrations. The solvent in the extracts is then removed and the residue taken up in a small amount of aqueous solution, thereby concentrating the analyte to a degree dependent upon the volume of reconstituting solution. Also, such solvents frequently have the effect noted above of freeing the analyte from endogenous binding proteins. The term "extraction" of a sample as used herein means extraction of sample analyte from the aqueous phase, with or without extraction of analyte bound to endogenous binding proteins or exclusion of substances related to the analyte of interest.
The organic solvents which have been employed for the above purposes are substantially water immiscible organic solvents, i.e. carbon-containing compounds which are liquid at one atmosphere pressure, partially or wholly within the range of greater than about 0.degree. C. and less than about 100.degree. C. Substantially water immiscible organic solvents are defined as organic solvents which are soluble in water at 20.degree. C. to the extent of less than about 15% v/v. Exemplary organic solvents that have been used heretofore for extraction include carbon tetrachloride, methylene chloride (dichloromethane), ethyl ether, ethyl acetate, petroleum ether, diethyl ether, ether, and benzene. Ordinarily the organic solvents are simply mixed with the sample, e.g., homogenized tissue, blood serum or urine, and incubated for a period sufficient to extract the desired substance. The organic layer is removed and evaporated, and the dry analyte then assayed.
Specific protein binding assays are frequently performed using insoluble surfaces physically coated with the binding proteins as opposed to the more difficult to manufacture covalently bound proteins. Such surface absorbed proteins are used in the assays to separate the protein bound analyte from that which remains in the liquid phase. One particularly popular technique is to absorb specific antibody onto the inner surface of a plastic test tube, usually polypropylene. See for example U.S. Pat. No. 3,646,346. Such test tubes are used in a typical competitive immunoassay by first adding aqueous solutions of the analyte and a labeled analogue of the analyte, or tracer, to a tube. After an incubation period to allow the tracer and analyte to compete for a limited number of protein binding sites, the protein-bound population of tracer and analyte is then separated from the population which remains in solution. This is conveniently done by decanting or aspirating the reaction solution from the tube.
The prior art has almost uniformly viewed the solvent extraction of samples to be a procedure separate and distinct from the specific binding assay. The practice of the prior art has been to completely remove the organic solvent employed in the sample extraction before the residual analyte is contacted with any of the reagents employed in the specific binding assay, e.g., antibody. Ordinarily the complete process has entailed removing the extract to a container, evaporating the solvent, resolubilizing the dry residue and then adding the reagents needed for the specific protein assay. This is clearly inconvenient, labor intensive and susceptible to considerable experimental error. However, the prior art has in at least one instance included organic solvent in the reagents used to perform a specific binding assay. According to U.S. Pat. No. 4,081,525, from about 0% to about 10% of a water miscible organic solvent can be used to extract steroids from carrier proteins present in samples while simultaneously conducting a specific protein binding assay. This method is of limited utility because, first, it provides for such a low concentration of organic solvent that the extraction efficiency may be unsatisfactory and, second, the solvent must be water miscible and therefore cannot be used to concentrate the sample analyte.